1. Field of the Invention
This invention relates to the isolation and purification of taxol and its natural analogues from a naturally occurring Taxus species or cell cultures thereof and, more particularly, to an improved method for isolating taxol and the congeners thereof from the Taxus species by reverse phase liquid chromatography.
2. Related Art
Taxol was first isolated in 1971 from the western yew, Taxus brevifolia by Wani et al. (1971), who characterized, its structure by chemical and X-Ray crystallographic methods.
Taxol is a member of the taxane family of diterpenes having the following structure: ##STR1##
Taxol and various taxane analogues or derivatives, including cephalomannine, are highly cytotoxic and possess strong in vivo activities in a number of leukemic and tumor systems. Especially, taxol is considered an exceptionally promising cancer chemotherapeutic agent. On the basis of its novel mode of action and activity shown in clinical trials, taxol was approved by the FDA for treatment of ovarian carcinoma. In view of the activity shown by taxol on other tumors such as breast and lung tumors, regulatory approval for the use of taxol in the treatment of these tumors is also currently under study. However, the major problem with the production of pharmaceuticals which incorporate taxol as an ingredient for treatments is the limited availability of the compound. One idea that has been thoroughly impressed on the minds of taxol scientists as well as on the lay public is that taxol is a very scarce commodity because the bark of the Pacific yew yields less than 0.01%.
A primary natural source for taxol is several species of very slow-growing yew (genus Taxus, family Taxaceae). The currently practiced procedures for isolating taxol from bark have the disadvantages of being fatal to the source, being very difficult to carry out, and producing low yields. For example, C.H.O. Huang et al. (1986) reported a 0.01% yield from a large scale isolation starting with 806 lbs. or more of Taxus brevifolia bark. Similar procedures have been reported which comparably produce low yields, ranging from as low as 0.004%, up to about (but not above) 0.017%. A yield of 0.01% translates into 1 g being isolated from 10 kg of the bark, or 1 kg of taxol from 10,000 kg (.apprxeq.22,000 lbs) of the bark. A mature tree is said to yield 20-25 lbs. of the bark, and this means that nearly 800-1000 trees are needed to produce a kilogram of taxol. Accordingly, use of the bark is being rapidly phased out as the primary source of taxol.
Senilh and co-workers studied the bark of the European yew (Taxus baccata) and described the isolation of a large number of compounds: taxol (or taxol A) (0.0165%), cephalomannine (or taxol B), 0.0064%), and others. The procedure used by Senilh and co-workers also includes multiple (seven) steps for the isolation of taxol, also primarily employing normal phase chromatography columns for the separation procedures.
(1) Extraction with alcohol and concentration. PA1 (2) Partition between water and dichloromethane. PA1 (3) "Filtration chromatography." PA1 (4) Silica column chromatography. PA1 (5) Alumina chromatography. PA1 (6) Medium pressure silica column chromatography. PA1 (7) Preparative HPLC. PA1 (1) The dried ground bark was extracted with methanol or ethanol and the combined extract concentrated to remove most of the alcohol. PA1 (2) The concentrate was then extracted with dichloromethane and the solvent extract concentrated to a powder. PA1 (3) This powder was stirred with a mixture of acetone and ligroin (1:1) and filtered to remove the insoluble matter. PA1 (4) The filtrate which contained taxol was concentrated, dissolved in 30% acetone in ligroin, and applied to a column of Florisil. PA1 (5) The taxol fraction from the column was purified by crystallization twice. PA1 (6) The crystalline taxol was further subjected to chromatography on a silica column. In this step, the closely related analogue, cephalomannine, was separated from taxol. PA1 (7) The purified taxol obtained from the column was crystallized twice. PA1 (8) Unseparated mixtures and mother liquors were recycled through the silica column to obtain additional amounts of pure taxol. PA1 (1) Extraction of the plant and concentration of the extract to a solid. PA1 (2) Defatting by partition between water and hexane. PA1 (3) Extraction with chloroform and concentration. PA1 (4) Silica column chromatography. PA1 (5) A second silica column chromatography. PA1 (6) Countercurrent distribution. PA1 (7) A second countercurrent distribution. PA1 (8) Preparative HPLC. PA1 (1) the fresh needles were extracted with 70% alcohol; PA1 (2) the extract was decolorized with charcoal and filtered; PA1 (3) the extract was concentrated to remove most of the organic solvent; PA1 (4) the aqueous concentrate was centrifuged to separate the precipitated solid (containing taxol); PA1 (5) the solid was then subjected to normal phase silica chromatography; PA1 (6) a second, low pressure silica column was run on the crude taxol fraction; and PA1 (7) a reverse phase column was used for final purification. Alternatively, instead of step (4), the aqueous concentrate was extracted with ethyl acetate, the extract concentrated, and applied to the silica column (step 5). By contrast, our process using a reverse phase column process which has particular advantages over normal phase chromatography, works well with the extract from the needles of this yew, even on a pilot plant scale. PA1 (a) treating the crude extract comprising taxol and its natural analogues by reverse phase liquid chromatography using a preparative scale column containing an adsorbent and causing the taxol and the natural analogues to be adsorbed on the adsorbent; PA1 (b) eluting the taxol and the natural analogues of taxol from the adsorbent; and PA1 (c) recovering the taxol and the natural analogues in separate fractions of the eluate.
For the other analogues, two or three other chromatographic columns, followed by preperative HPLC, were used.
Other schemes for the large-scale production of taxol from T. brevifolia bark have also been developed. One such method used by Polysciences, Inc. includes the following steps:
The yield of taxol in this process was reported as 0.004-0.01% based on the bark used. The isolation was described by other workers: Miller et al., 1981; McLaughlin et al., 1981; Kingston et al., 1982; and Senihl et al., 1984. The reported yields of taxol from various species of yew range from 50 mg/kg to 165 mg/kg (i.e., 0.005-0.017%). At present, the bark of Taxus brevifolia is still being used as the major source of taxol.
Because of (a) the rather low (0.01% or less) yields of taxol from the bark, (b) the relative unavailability of any other useful analogues, and (c) the need to cut the slow-growing trees to harvest the bark, it was decided that the bark was not an attractive source for taxol. Therefore, besides isolation from the bark, there are currently three avenues that are being pursued for the future production of taxol: (1) isolation from renewable plant parts, e.g., the ornamental yew clippings and needles; (2) semi-synthesis of taxol; and (3) production of taxol by tissue culture procedures.
Laboratory scale isolations of taxol and its analogues from different parts of different species of Taxus have been described. Miller and co-workers (see Miller et al. [1981], supra), working with the roots, stems, and leaves of Taxus wallichiana, isolated taxol (0.01%), an analogue called cephalomannine (0.016%), among other now-commonly known analogues. The Miller procedure consists of eight steps, including two normal phase chromatography steps.
Kingston and co-workers (Kingston, et al, 1982), working with taxol-free fractions obtained from the large-scale processing of the bark of Taxus brevifolia, isolated very minute yields (&lt;0.0003% ) of these analogues, which reinforce the notion that the yield of taxol from T. brevifolia is very low and that few, if any, useful analogues can be obtained. From the needles of Taxus baccata, Colin et al. isolated 10-deacetyl baccatin III, which they used to synthesize a number of derivatives of taxol. See U.S. Pat. No. 4,814,470.
The use of the tissue from the ornamental yew (Taxus x media Hicksii) for isolating taxol and taxanes has been described in U.S. Pat. No. 5,279,949. The process described in the '949 patent, however, involves column chromatography using normal phase silica. Specifically, the '949 patent describes a separation procedure as follows:
Upon the discovery of Taxus x media Hicksii needles as a taxol source, Witherup et al., 1990 showed that the needles contain as much taxol as the bark, i.e., about 0.01%, and isolated taxol by an unspecified method in a yield of 0.006%. This plant produces several unrelated taxanes which follow taxol in the purification step (whether using the normal phase or reverse phase column method) and their complete removal from taxol (to meet the FDA specifications) will require at least two (or more) columns besides the initial column. Unlike the situation with cephalomannine, which is present only to a minor extent when taxol is isolated from bark, the taxanes that accompany taxol isolated from the needles of the ornamental yew are present in much higher concentrations than taxol.
The published literature on this subject generally consists of methods using analytical HPLC of the needles (and other parts of the plants) and listing the yields based on these analyses. It is also clear from some of the papers that the needles contain, besides taxol, some unrelated taxaries (cinnamate esters with a 4/20 double bond instead of the oxetane ring) which co-elute with taxol in the analytical HPLC (Castor and Taylor, 1993). Two of these compounds were isolated in impure form and characterized spectrally (Chmurny et al., 1993).
Also, in view of the high therapeutic potential of taxol, the synthesis of the compound has attracted much interest among synthetic chemists worldwide. Although methods for total synthesis of taxol have been announced by at least two groups of researchers, development of a practical process is likely to be several years away. The semi-synthesis procedure involves conversion of a taxol precursor to taxol through a series of several chemical conversion steps. The European yew, Taxus baccata, is being cultivated on a large scale for the isolation of 10-deacetyl baccatin III from its needles, so that tiffs compound can be converted into taxol through semisynthesis.
The yield of 10-deacetyl baccatin III from the needles of T. baccata is variably reported from 0.02-0.1%, with an average of 0.05 %. It appears that neither taxol nor any of the other analogues are being isolated from this source. In addition, the semisynthetic conversion is said to involve seven steps and, under the best of circumstances (90% yield at each step), an overall yield of 40% may be expected. This translates into a relatively low yield of approximately 0.02% from the plant source.
The subject process can be applied to the isolation of 10-deacetyl baccatin III, as well as taxol and other analogues, from the needles of T. baccata.
Much progress has been made over the past few years to grow the Taxus callus tissue under cell culture conditions to produce taxol. It is widely assumed that this method may replace others that are based on conventional plant extraction, etc. Indications are that the culture produces not only taxol, but also cephalomannine and some of the xylosides. Therefore, a simple, inexpensive purification procedure will still be necessary if such tissue culture methods are ultimately developed for wide-scale use.
Because of the current state of available synthesis procedures, and other alternative methods for purifying or obtaining the valuable taxol compound, the isolation of taxol from Taxus species, despite low yields, will be the only reliable supply source for clinical quantities of taxol for years to come. Unfortunately, the currently available isolation methods require multiple steps, which translates into increased time and expense while still producing relatively low yields. Thus, simplified purification techniques which provide higher yields of taxol are needed to provide greater quantities of this promising therapeutic agent at reduced cost. The present invention provides a purification technique which accomplishes this goal.